Objective:To analyze the distribution of different SF3B1 genotypes in patients with myelodysplastic syndromes (MDS) and its prognostic value.Methods:Totally, 377MDS patients who were initially diagnosed in the First Affiliated Hospital of Nanjing Medical University from January 2014 to January 2022 were included in the retrospective analysis.The patients were divided into three different groups according to mutation stcote of SF3B1, including 317 patients with SF3B1 wild type (SF3B1 WT) (214 males and 103 females, 63(49, 71) years old),39 patients with SF3B1 K700E mutation(SF3B1 K700E(17 males and 22 females, 65(52, 73)years old)) and 21 patients with SF3B1 non-K700E mutation(SF3B1 non-K700E)(13 males and 8 females, 67(63, 73) years old). MDS-related 20 gene mutations were detected using targeted sequencing technology; Survival curves were constructed by the Kaplan-Meier method; Cox proportional hazards model was established to evaluate different factors at diagnosis on survival by univariate and multivariate analyses.. Results:Compared with SF3B1 non-K700E patients, SF3B1 K700E patients had a higher median absolute neutrophil count ( P=0.002) and were likely to be in the low/int-1 International Prognostic Scoring System (IPSS) categories ( P=0.023). A 20-gene targeted sequencing analysis showed that, compared with SF3B1 WT patients, SF3B1 K700E patients were associated with lower frequency of ASXL1 and U2AF1 mutations ( P=0.018 and P=0.003); while compared with SF3B1 non-K700E patients, the frequency of ASXL1 mutation was significantly lower in SF3B1 K700E cases ( P=0.029). Patients with SF3B1 K700E had better overall survival (OS) in comparison with SF3B1 WT and SF3B1 non-K700E in MDS patients ( P<0.001 and P=0.045, respectively). In comparison with SF3B1 WT patients, SF3B1 MUT patients had more favorable OS and progression-free survival (PFS) in MDS without excess blasts ( P<0.001 and P<0.001, respectively), but no significant difference was found in MDS with excess blasts ( P>0.05). Compared with SF3B1 WT patients, SF3B1 K700E patients had superior OS and PFS in the int-1 IPSS category ( P=0.010 and P=0.013, respectively). By multivariable analysis, the presence of SF3B1 K700Ewas an independent predictor of superior OS ( HR=0.461,95% CI 0.262-0.811, P=0.007). Conclusion:SF3B1 K700E and SF3B1 non-K700E patients had significantly improved OS in comparison with SF3B1 WT MDS patients. Furthermore, SF3B1 K700E patients were associated with a better OS compared with SF3B1 non-K700E MDS patients. SF3B1 mutation could not overcome the poor prognostic effect of excess blasts, which highlights the importance of the SF3B1 mutation subtype in risk assessment of MDS without excess blasts.

The main purpose of our study was to evaluate the efficacy and safety of eltrombopag plus cyclosporine A (CsA) in transfusion-dependent non-severe aplastic anemia(TD-NSAA). The clinical characteristics of 13 TD-NSAA patients who received initial treatment of eltrombopag plus CsA from 2019 to 2021 were retrospectively analyzed. The 3-month overall hematological response (OR) rate was 12/13. Until the end of follow-up, 12 patients responded, among whom 2 patients reached complete response (CR) and 9 patients reached partial response (PR) and 1 with HR. Paroxysmal nocturnal hemoglobinuria (PNH) developed in one patient at 6 months after treatment. Five of thirteen patients reported mild adverse reactions, which were all manageable. Compared with historical data, the combination of eltrombopag with CsA is an effective regimen in patients with TD-NSAA.

Objective:To investigate the clinical significance of cc-chemokine receptor 7 (CCR7) as a potential diagnostic or differential marker for chronic lymphocytic leukemia (CLL).Methods:A total number of 643 patients with B-cell chronic lymphoproliferative diseases (B-CLPD) admitted to the First Affiliated Hospital of Nanjing Medical University from January 2015 to December 2018 were enrolled. The patients included 327 cases of CLL, 58 cases of mantle cell lymphoma (MCL), 34 cases of follicular lymphoma (FL), 36 cases of marginal zone lymphoma (MZL), 10 cases of hair-cell leukemia or its variants (HCL/HCLV-v), 40 cases of Waldorf′s macroglobulinemia (WM), 48 cases of CD5 +B-cell chronic lymphoproliferative disease unclassified (B-CLPD-U) and 90 cases of CD5 -B-CLPD-U. At the same time, 20 samples from healthy people from the medical examination center of our hospital were used as normal controls. Flow cytometry was used to detect the immune-phenotype and CCR7 expression level in B-CLPD patients, and Fluorescence in situ hybridization (FISH) was used to analyze the genomic alterations: the ataxia telangiectasia mutant gene (ATM) deletion, the 13q14 deletion, the P53 deletion and trisomy 12. Sanger sequencing was used to analyze gene mutations of splicing factor 3B subunit 1 (SF3B1), NOTCH1, tumor protein 53 (TP53) and immunoglobulin heavy chain variable region (IGHV). Measurement data were compared by Mann-Whitney test, and the positive rates were compared by chi-square test. The diagnostic value and optimal positive cutoff value of CCR7 were calculated using receiver operating characteristic (ROC) curve. Results:The positive rates of CCR7 expression in typical CLL and atypical CLL were 90.8% (257/283) and 84.1% (37/44), respectively, and there was no significant difference of the positive rates (χ 2=1.228, P=0.268) between groups. The positive expression rates of CCR7 in CLL, MCL, CD5 +B-CLPD-U, CD5 -B-CLPD-U, FL, WM, HCL/HCL-v and MZL were 89.9% (294/327), 10.3% (6/58), 6.3% (3/48), 8.9% (8/90), 0, 0, 0 and 13.9% (5/36) respectively, and the median mean fluorescence intensity (MFI) was 278 (246, 307), 114 (106, 128), 112 (106, 117), 110 (104, 121), 108 (105, 119), 111 (105, 124), 112 (108, 115) and 109 (105, 120) respectively. Compared with CLL, the positive expression rates of CCR7 in other types of B-CLPDs were lower significantly (χ 2=181.3, 177.8, 232, 164.7, 180.8, 62.6, 129, P<0.01). In addition, the sensitivity, specificity and accuracy of CCR7 for distinguishing CLL from other types of B-CLPD were 89.9%, 93.0% and 92.3%, respectively. The positive expression rate of CD49d in CCR7 +CLL patients was 13.9%, which was significantly lower than that in CCR7 -CLL patients (42.1%) (χ 2=7.6, P=0.01). The coincidence rate of 13q14 deletion was 50.3% in CCR7 +CLL patients, which was significantly higher than that in CCR7 -CLL patients (20%) (χ 2=6.56, P=0.01). Conclusions:The CC-chemokine receptor 7 (CCR7) antigen is an effective marker for the diagnosis and identification of chronic lymphocytic leukemia (CLL). The expression level of CCR7 in clinical specimens can distinguish CLL from other pathological subtypes of B-CLPDs.

Objective:To explore the correlation of CD49d expression patterns with molecular genetics and hotspot gene mutants in patients with chronic lymphocytic leukemia.Methods:The expression of CD49d was detected by flow cytometry and grouped into homogeneous, bimodal, negative and positive expression. Panel fluorescence in situ hybridization (FISH) was used for molecular genetics analysis and next-generation sequencing (NGS) was conducted for gene mutation detection.Results:There were 43 patients (23.89% ) with positive CD49d expression, 137 patients (76.11% ) with negative CD49d expression, 96 patients (53.33% ) with homogeneous CD49d expression and 84 patients (46.67% ) with bimodal CD49d expression. Compared with patients in the CD49d negative group, patients in the CD49d positive group had higher Rai stage ( P=0.048) and higher proportion of spleen enlargement ( P=0.030) . Compared with patients with homogeneous expression of CD49d, patients with bimodal expression of CD49d had a higher proportion of spleen enlargement ( P=0.009) . The expression rate of 11q22- in bimodal CD49d - group was significantly higher than that in homogeneous CD49d - group (24.29% vs 10.45% , P=0.043) . The incidence of +12 in homogeneous CD49d group was higher than that in bimodal CD49d group (16.67% vs 5.95% , P=0.035) . The incidence of +12 in homogeneous CD49d + group was higher than that in bimodal CD49d - group (17.24% vs 4.29% , P=0.045) . The incidence of +12 in homogeneous CD49d - group was higher than that in bimodal CD49d - group (16.42% vs 4.29% , P=0.024) . BIRC3 mutation rate in CD49d positive group was higher than that in CD49d negative group (11.63% vs 2.92% , P=0.037) . Conclusion:There were significant correlations between CD49d and 11q22-, +12 and BIRC3 gene mutation. Patients with bimodal CD49d were more correlated with poor prognosis indexes.

Objective:To analyze the differences in immunophenotype, cytogenetics, and molecular biology between typical and atypical immunophenotype chronic lymphocytic leukemia (CLL) , and explore the correlation of cytogenetic anomalies with gene mutations.Methods:This study included 488 patients diagnosed in the First Affiliated Hospital of Nanjing Medical University between November 2014 and May 2021. Of these, 382 patients scored 4-5 points, which was typical CLL (tCLL) , and 106 scored 3 points, which was atypical CLL (aCLL) as per the Royal Marsden Hospital Immunomarker Integral System. Peripheral blood cells were collected for immunophenotype by multiparameter flow cytometry in 488 patients, fluorescence in situ hybridization (FISH) was employed to detect cytogenetic anomalies in 359 patients, and gene mutations were detected by next-generation sequencing (NGS) in 330 patients.Results:The positive rates of CD10, CD22, CD49d, CD81, and FMC7 were significantly higher in the aCLL compared with the tCLL group ( P=0.020, P<0.001, P<0.001, P=0.027, and P<0.001, respectively) , while the positive rates of CD5, CD23, CD148, and CD200 were lower in the former compared to the latter ( P<0.001, P=0.017, P=0.041, and P<0.001, respectively) . aCLL exhibited a higher frequency of trisomy 12 and lower frequency of del (13q14) compared to the tCLL group ( P<0.001 and P<0.001, respectively) . Moreover, aCLL patients also showed a higher incidence of NOTCH1 mutations than the tCLL patients ( P=0.038) , while no statistically significant differences in other gene mutations occurred between the two groups. No significant differences in overall survival (OS) and treatment-free survival (TFS) occurred between aCLL and tCLL using Kaplan-Meier analysis ( P>0.05) . Conclusion:aCLL has characteristic immunophenotype, cytogenetic, and somatic mutation that differ from tCLL, and this can provide reliable information for the diagnosis and differential diagnosis between the two groups.

Objective:To investigate the expression characteristics of TRBC1 protein in mature T-cell lymphoma (TCL) , and compare with T-cell receptor (TCR) -Vβ repertoire analysis and TCR gene rearrangement results, to explore the value of TRBC1 in the diagnosis of TCL.Methods:The expression of TRBC1 was detected by multi-parameter flow cytometry in 30 cases of TCL, 40 cases of normal controls and 50 cases of patients without T lymphocyte proliferative diseases (non-TCL) admitted to the Department of Hematology, The First Affiliated Hospital of Nanjing Medical University. The diagnostic value of TCRVβ repertoire analysis, TCR gene rearrangement and TRBC1 restricted expression detection in TCL was evaluated.Results:The positive rates of CD4 +T and CD8 +T cell subsets TRBC1 in normal control group were (39.6±6.5) % and (39.3±4.4) %. The positive rates of CD4 +T and CD8 +T cell subsets TRBC1 in non-TCL were (39.1±3.8) % and (36.0±8.4) %. All 30 cases of TCL were CD3 +TCRγδ -, and the positive rate of TRBC1 was >92.3% or <12.7%. All cases showed restrictive expression pattern (monoclonal expression) , which was significantly different from those of the normal control and the non-TCL cases ( P<0.001) . In terms of the diagnostic performance of T cell clonality, the sensitivity of TRBC1 was 100%, the positive detection rate of TCR gene rearrangement was 92.8%, and the sensitivity of TCRVβ detection was 94.1%. Kappa test showed high consistency among the three detective methods. Conclusion:Multi-parameter flow cytometry detection of TRBC1 expression level can quickly and efficiently diagnose mature T-cell lymphoma, which has good clinical application value.

Objective:The clinical characteristics and prognosis of 20 patients with small B-lymphocyte proliferative disease with t (14;19) (q32; q13) were analyzed to improve the understanding of such rare cases.Methods:The clinical data of 20 patients with t (14; 19) (q32; q13) small B lymphocyte proliferative disease treated in the First Affiliated Hospital of Nanjing Medical University from April 2013 to December 2020 were retrospectively collected and analyzed. Among them, 10 cases were chronic lymphocytic leukemia (CLL) and 10 cases were other small B-cell malignancies.Results:Among the 20 cases, 10 were male and 10 were female, and the median age at diagnosis was 53.5 (35-88) years old. All patients had absolute lymphocytosis, 19 patients had lymphadenopathy, and 10 patients had splenomegaly. With a median follow-up of 36 (4-163) months, three patients died, and 11 patients had a time to treatment (TTT) ≤12 months. Ten patients (50%) were accompanied by +12, two patients (2/17, 12%) were accompanied by 13q-. Moreover, we found that t (14;19) was associated with unmutated immunoglobulin heavy-chain variable (IGHV) somatic mutation (17/19, 89%) and a biased use of IGHV4-39 (7/17, 41%) was observed. Next-generation sequencing detected one or more gene mutations in 14 (14/17, 82%) cases and a total of 25 gene mutations had been revealed, of which the most frequent were NOTCH1 (35%) , followed by SF3B1 (24%) and KMT2D (18%) . For 10 CLL patients, five (50%) were defined as Rai Ⅲ/Binet C. It is noteworthy that among the 20 cases, two cases actually involved Richter transformation.Conclusions:Small B-cell malignant tumors with abnormal t (14; 19) show unique clinical biological characteristics, often accompanied by a variety of adverse prognostic factors, and tend to have an aggressive clinical course.

Objective:To investigate the clinical, genetic, and clonality related aspects of individuals with Richter transformation (RT) .Methods:From January 2019 to December 2021, 18 RT patients with diagnoses at the First Affiliated Hospital of Nanjing Medical University (Pukou CLL center) were retrospectively examined. The immunoglobin heavy variable (IGHV) gene usage and IGHV-D-J rearrangement pattern of diagnosed CLL/SLL and transformed diffuse large B-cell lymphoma (DLBCL) were compared to determine the clonality relatedness. To investigate the risk factors of RT, Clinical and laboratory data from patients with newly diagnosed CLL/SLL and transformed DLBCL were gathered.Results:The median age of RT was 56.5 (41-75) years old. 17 patients transformed to DLBCL and 1 transformed to Hodgkin lymphoma (HL) . Of 17 individuals who had DLBCL transformation, 15 had CLL/SLL-related clonality and 2 had unrelated clonality. Next-generation sequencing (NGS) analysis of 11 paired initially diagnosed treatment-naive CLL/SLL and RT DLBCL found that EGR2、TP53 and NOTCH1 were among the most frequently mutated genes both in treatment-naive CLL/SLL and in RT DLBCL. In several cases, specific mutations were gained or lost throughout RT, indicating clonal evolution. Among 10 patients before exposure to BTK inhibitors before RT, four patients acquired BTK mutation. The aforementioned mutations should be considered high-risk variables for transformation; in addition, TP53 and EGR2 mutations could be linked to a poor prognosis following RT in patients receiving a cocktail of new medicines.Conclusion:Most RT DLBCL patients in our center are clonality related (15/17, 88.2% ) and we recommend all qualified centers to evaluate clonality relatedness of RT DLBCL patients. There was some variability in the mutational landscape between DLBCL that had undergone a transformation and initially diagnosed, treatment-naive CLL/SLL. The underlying molecular mechanism of RT needs more research.

Objective:To investigate the expression pattern of TCR variable region subfamily (Vβ and Vδ) in patients with mature T-cell lymphoma (TCL), and to compare the diagnostic value of TCRVβ and TCRVδ analysis in TCL.Methods:The TCRVβ flow cytometry kit was used to detect the expression of Vβ subtypes of αβT cell in 199 patients with αβ TCL and 398 patients with non-TCL, who hospitalized in Jiangsu Provincial People Hospital from 2011 to 2020. Among them, 185 cases of αβ TCL and 355 cases of non-TCL also underwent TCRβ and TCRγ gene rearrangement detection. The TCRVδ based 10-color protocol was used to detect the expression of Vδ subtypes in 24 cases of γδTCL, 10 cases of normal controls, and 15 cases with reactively higher CD4 and CD8 double-negative ratio from 2017 to 2020, and 24 cases of γδTCL and 15 cases with reactively higher CD4 and CD8 double-negative ratio underwent TCRβ, TCRγ and TCRδ gene rearrangement detection. The diagnostic performance and degree of coincidence for detecting malignant clonality were compared between TCRVβ and TCRVδ analysis and the TCR gene scanning method.Results:In the 199 cases of αβ TCL, 182 cases (91.5%) showed restricted expression or the sum of the positive percentages of the subgroups was less than 30% for the 24 TCRVβ subtypes. Among them, the subfamily members with the highest incidence of clonal T lymphocytes were TCRVβ13.2 (12.6%, 23/182) and TCRVβ3 (8.2%,15/182); the TCRVβ subtypes showed nonclonal results in 99.0% (394/398) of non-TCL. All 24 cases of γδTCL (100%) showed abnormal distribution patterns of Vδ1 and Vδ2, of which 19 cases showed restricted expression of Vδ1, and the remaining 5 cases had negative expression of either Vδ1 or Vδ2, and the positive rate of Vδ1 cells was significantly higher than that of Vδ2 cells (79.9%±10.8% vs 0.7%±0.3%, P<0.001). Among the normal control and cases with reactively higher CD4 and CD8 double-negative ratio, the positive rate of Vδ2 cells was significantly higher than that of Vδ1 cells (73.7%±6.7% vs 15.6%±4.2%, P<0.001), and all cases (25/25) showed a normal distribution pattern. In terms of the diagnostic performance of TCL, there was no significant difference of sensitivity and specificity between TCR variable region subfamily detection by flow cytometry and TCR gene scanning technology (the sensitivity was 92.4% and 91.4% respectively; the specificity was 99.0% and 95.9% respectively, P=0.065), and the coincidence rate of the two diagnostic methods is high (Kappa=0.809, P<0.001). Conclusion:Detection of TCR variable region subfamily by flow cytometry could quickly and effectively diagnose mature TCL.

Objective:This study aims to explore the clinical characteristics of T-cell large granular lymphocyte leukemia (T-LGLL) patients with STAT3 mutation status and provide a reference for clinical management of such patients.Methods:The clinical data of T-LGLL patients between 2009 and 2019 in Jiangsu Province Hospital were retrospectively analyzed. Differences in baseline clinical data, treatment responses, and survival outcomes in patients with STAT3 mutations or with no mutations were compared.Results:A total of 80 patients were included, including 66 patients without STAT3 mutation and 14 patients (17.5%) with STAT3 mutation. The frequency of Y640F mutation was the highest (42.9%) . Compared with non STAT3 mutation group, STAT3 mutation group had lower HGB (67.5 g/L vs 82.5 g/L, P=0.018) , lower neutrophil count (0.665×10 9/L vs 1.465×10 9/L, P<0.001) , higher LDH (229 U/L vs 198 U/L, P=0.041) , higher ferritin (402.5 g/L vs 236.0 g/L, P=0.029) , higher expression rate of TCR Vβ subfamily (89.2% vs 65.4%, P=0.014) and higher proportion of patients with treatment indications (100% vs 74%, P=0.033) . The complete remission rates of STAT3 mutation group and non mutation group were 38.5% and 32.7%, respectively, with no significant difference ( P=0.748) . The overall response rate of first-line immunosuppressive therapy in STAT3 mutation group and non mutation group were 69.2% and 69.4%, respectively, with no significant difference ( P=1.000) . The median follow-up time was 63 (2-121) months. There was no significant difference in the overall survival time between the two groups ( P=0.170) . Conclusions:T-LGLL patients with STAT3 mutations seems to be correlated with an increased tumor burden and high treatment demand, and had a good response to first-line immunotherapies. The prognostic significance of STAT3 mutation in T-LGLL patients requires further validation.